SUMMARY Despite considerable clinical impact of blocking checkpoints such as PD1 and LAG3, the mechanisms remain poorly understood. One remaining gap is how PD1 and LAG3 regulate formation of TEX precursors early in chronic infection and mature TEX subsets later. Answering this question will identify molecular mechanisms that link PD1 and LAG3 to preventing or reversing exhaustion and new therapeutic opportunities. We hypothesize that individual and combined signals from PD1 and LAG3 engage temporally distinct, therapeutically relevant mechanisms to regulate T cell exhaustion that will be discovered by dissecting the synergy between these pathways at different stages of T cell exhaustion. This project will address this hypothesis by testing: Aim 1: How do signals from PD1 or LAG3 or both promote formation of TEX precursors? Here, we will reveal how PD1 and/or LAG3 are involved in initial molecular and cellular establishment of early TEX formation and provide opportunities for preventing development of exhaustion. We hypothesize that PD1 and/or LAG3 are necessary to initiate and temporally reinforce development of TEX during chronic viral infection through mechanisms that involve TCF1, NFAT, and/or TOX. We will use constitutive or inducible CD8 T cell-intrinsic PD1 and/or LAG3 deficiency together with antibody (Ab) blockade and novel exhaustion tracking mice (i.e.Lag3CreERT2.Rosa26LSL.tdTomato or ToxCreERT2.Rosa26LSL.tdTomato) from Core B. Thus, Aim 1 will deliver detailed maps of how PD1 and LAG3 separately and together regulate initial formation of TEX. Aim 2: What are the molecular and epigenetic events caused by temporally induced loss of PD1 or LAG3 in mature TEX subsets? Despite the clinical relevance of checkpoint blockade, the underlying biology of TEX reinvigoration remains poorly understood, particularly surrounding early molecular events associated with reinvigoration in vivo and its impact on different TEX subsets. We hypothesize that early molecular events following removal of PD1 and/or LAG3 are distinct for different TEX subsets, imparting novel functional, transcriptional, and/or differentiation changes that will enable us to identify new molecular targets to reverse or prevent exhaustion. Here, we will interrogate bulk and single-cell transcriptional as well as epigenetic changes in total TEX and TEX subsets over a high-resolution time-course following removal of PD1 and/or LAG3. Discoveries will be further dissected using in vivo CRISPR/Cas9 screening and RV approaches (Core C). These data will provide important insights for applying PD1 and/or LAG3 blockade in humans. PPG Interactions: Because the core program of exhaustion is conserved in chronic infections, tumors, and autoimmunity, Project 3 will connect extensively with Projects 1 and 2 for experimental models and mechanistic insights from autoimmunity or tumors. We will also coordinate with Core A to exchange data and interact, Core B to obtain mice, Core C for RV approaches and bioinformatics, and Core D for immunohistology.